This project is divided into two parts, in vivo and in vitro. For the in vivo studies, pregnant monkeys are being treated from day 30 to day 132 with 1 mg/kg nicotine per day. These studies have been completed and analyses are ongoing. The in vitro studies involved fetal monkey lung organ culture. One such study has been performed to date. For this study, 90 day fetal monkey lung was incubated for 3 or 5 days in 0, 100 nM, 1 fM or 5 fM nicotine q 10 nM GRP. (Based on the literature the best estimate for nicotine concentrations in amniotic fluid is 20 - 300 nM depending on time from last cigarette.) Effects of nicotine were quantified by lung morphometry, measurement of mitotic index (BrdU uptake), and immunohistochemical and in situ hybridization detection of GRP and GRP receptor expression. In situ analyses are still ongoing, but morphometry and labeling studies have yielded potentially very significant results. 100 nM nicotine caused a significant decrease in airway epithelial labeling, but a non-significant decrease in mesenchymal labeling. Consistent with this, the epithelial volume density and the ratio of epithelial to mesenchymal volume densities was decreased in nicotine treated lung. By contrast, GRP had an almost opposite effect, stimulating mitogenesis of epithelial cells (as we have previously reported; Li et al., J Clin Invest 1994; 94:1605-1615) with little effect on mesenchymal cells. Remarkably, nicotine completely blocked the effects of GRP. The next step will be to determine the mechanism of nicotine's action and the mechanism mediating the interaction between GRP and nicotine. The first steps in this process will be to determine which nicotinic receptor subunits are expressed in developing lung, which cells in lung express the nicotinic receptor, and what the effect of nicotine on GRP receptor expression is. These findings are highly relevant to the hundreds of thousands of human infants born every year who are exposed to nicotine in utero.